This proposal is a response to RFA AI-10-010 requesting applications among others to develop antimicrobial agents against Clostridium difficile. C. difficile, a serious nosocomial cause of diarrhea and intestinal inflammatory disease, has increased significantly in both North America and Europe, causing lengthy hospitalization and substantial morbidity and mortality. Many patients who initially appear to have been cured suffer multiple relapses. While other interventions have been tried (probiotics, toxin-absorbing polymers, and toxoid vaccines), neither prevention nor treatment strategies have kept up with the increasing numbers of incidence of CDI, due in part to the epidemic strain, NAP-1/027, that produces larger quantities of toxins A and B and carries the binary toxin CDT, whose function is unclear. Vancomycin, the first drug to be used remains the most widely used and the only FDA approved drug, although metronidazole is frequently prescribed. The goal is to develop a new class of antibacterials for treatment of CDI. MBX 259C, the prototype drug, is a dual acting DNA replication inhibitor which targets DNA polymerase through its anilino uracil (AU) component and gyrase/topoisomerase through its fluoroquinolone (FQ) component. In preliminary studies, this compound has both low oral bioavailability and a narrow spectrum of activity. It was found to specifically inhibit C. difficile, including the NAP-1/027 fluoroquinolone-resistant strains (MIC90 = 2 &#956;g/mL), without affecting most anaerobic intestinal bacteria. MBX 259C was found to protect hamsters and mice from both acute CDI. The poor oral absorption of MBX 259C should maximize its gut concentration and minimize any systemic toxicity when dosed orally. For this work, we have developed the antibiotic-treated mouse model for screening analogs generated by chemistry, and effective inhibitors will then be tested in the chronic pseudomembranous colitis (PC) piglet model we have described recently (Steele et al 2010) for preclinical evaluation. More specifically we will: Specific Aim 1: Synthesize and optimize a library of MBX 259C hybrid analogs for in vitro potency testing against clinical isolates of C. difficile, with a view to, a) increase solubility and MIC in vitro potency (&#8804;0.5&#956;g/mL), b) increase bactericidal activity and inhibition of sporulation, and c) minimizing impact on other gut flora. Specific Aims 2&3: Characterize MBX 259C and newly synthesized analogs in the infection mouse model to establishing the optimal effective dose in relation to frequency of drug administration and duration of treatment. Specific Aim 4: Select two top ranking hybrid analogs, screened in mice, for preclinical evaluation in the diarrhea piglet model, including effective delivery (capsular formulation) of drugs to the large bowel. Specific Aim 5: Conduct PK/Tox animal studies to establish a safety margin and determine systemic/oral doses. In five years we will have one or more well characterized compounds that have been preclinically evaluated in two animal models for safety and efficacy, with a well established dose and duration of administration, packed into a suitable capsule, ready for IND submission and safety evaluation in phase I.